A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens

Abstract Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1‐based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1‐based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1‐based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B‐based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.

growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant.
Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens. Methylorubrum extorquens has great potential to become a universal production strain for the C1-based bioeconomy (Chen & Lan, 2020;Ochsner et al., 2014;Zhang et al., 2019). In the last decade, several M. extorquens strains with heterologously expressed metabolic pathways were described for the biotechnological production of various products including 1-butanol, 3-hydroxypropionic acid, mono-and dicarboxylic acids, mevalonate or α-humulene (Hu & Lidstrom, 2014;Liang et al., 2017;Lim et al., 2019;Schada von Borzyskowski et al., 2018;Sonntag et al., 2014, Sonntag, Kroner, et al., 2015Sonntag, Müller, et al., 2015;Yang et al., 2017). The development of production strains, however, is limited by the organism's MicrobiologyOpen. 2022;11:e1325. www.MicrobiologyOpen.com restricted genetic accessibility. Although the bacterium has served as a model organism for methylotrophy for many decades and is therefore well described, only a few genetic tools have been developed. Plasmids are a key tool for the biotechnological applicability of a strain. The simultaneous use of two independent plasmids provides experimental flexibility and simplifies screenings and production strain developments. The compatibility of two or more plasmids in a single bacterial cell requires not only different selection markers but, more importantly, different origins of replication and partitioning systems (del Solar et al., 1998;Novick, 1987). As part of their characterization, plasmids are divided into Inc groups: plasmids from the same group are usually incompatible.
For M. extorquens AM1, besides a recently described set of mini chromosomes (Carrillo et al., 2019), derivatives of the pCM plasmid system (Marx & Lidstrom, 2001) were used almost exclusively as episomal vectors for heterologous gene expression.
The expression vectors pCM80 (tc R ) and pCM160 (kan R ) are based on pDN19, a small IncP vector (Marx & Lidstrom, 2001). The authors of the respective study isolated the derivative pDN19X from M. extorquens AM1 transformed with pDN19. This derivative could be efficiently maintained and retransferred in M. extorquens AM1 and was used as a basis for the development of the pCM system (Marx & Lidstrom, 2001). In their work, the authors also showed that pBBR1MCS-2, a vector derived from Bordetella bronchiseptica pBBR1, could be transferred into M. extorquens with very low transformation efficiency and a significantly reduced transformant growth rate. Since this vector is known to be compatible with IncP group plasmids (Antoine & Locht, 1992;Kovach et al., 1994Kovach et al., , 1995, we chose it as a starting point for the development of a plasmid with compatibility with the pCM vector system. The GC content of 64.6% seems to be applicable to M. extorquens AM1 (overall GC content of 68.5% [Vuilleumier et al., 2009]) and its diverse usability as a broad range vector can be also useful for certain applications. Here, we describe a pBBR1-derived plasmid with a mutation upstream of the rep gene that confers suitability for cotransformation with established pCM plasmids.

| Plasmids and bacterial strains
A list of all used plasmids and bacterial strains is given in Table 1.
Reporter gene plasmids were constructed by subcloning synthesized DNA fragments (BioCat) into plasmid backbones. Used sequences and restriction enzymes are listed in Table 2. Analysis of sequences and creation of plasmid map was done with SnapGene (www. snapgene.com). Transformation of M. extorquens AM1 with plasmid DNA was performed as previously described (Toyama et al., 1998).
If not stated differently, 100 ng of plasmid DNA was used for transformations.
The amount of starting material for the final experiments was 500 pg of template DNA. All reactions were performed in biological triplicates. The relative ratio (R) was calculated with the Pfaffl efficiency-corrected model with averaged controls (Equation 2): (Pfaffl, 2004).
T A B L E 3 Primers used for real-time polymerase chain reactions F I G U R E 1 Growth of Methylorubrum extorquens AM1 after transformation with pCM160 or pBBR derivatives and DNA sequence differences between the used pBBR derivatives pMis1 and pMis1_1B. (a) Transformation plates after transformation of M. extorquens AM1 with 30 fmol of pMis1, pMis1_1B, or pCM160. Transformation plates were photographed after 96, 120, and 240 h of incubation. (b) Mutation in the sequence of pMis1_1B. The inserted nucleotide (cytosine) is located 22 bp upstream of the rep gene, whose start codon is indicated by italic letters.
Consequently, modifications in the rep gene region have been shown to influence the PCN (Mi et al., 2016;Tao et al., 2005;Wadood et al., 1997). Interestingly, the currently used pCM system is based on a similar experiment with the plasmid pDN19 (Marx & Lidstrom, 2001).

| Compatibility of pMis1_1B with the established pCM plasmids
For testing the compatibility of pMis1_1B with the pCM system, M.
extorquens AM1 was transformed with pMis1_1B and pCM80 simultaneously. The cotransformation did lower the transformation efficiency by 2-fold compared to transformation of pMis1_1B alone ( Figure 2a). Furthermore, colonies of cotransformants were only clearly visible after 8 days of growth, indicating an increased metabolic burden caused by the presence of both plasmids. Detailed growth monitoring in liquid growth medium confirmed these results: While M. extorquens AM1 harboring pMis1_1B already showed a reduced growth rate compared to the pCM160-containing strain or the plasmid-free strain, cotransformation with both plasmids strongly reduced the growth rate (Figure 2b). Plasmid-induced reduction of growth rates under methylotrophic growth conditions due to metabolite limitations have already been described for M. extorquens AM1 (Kiefer et al., 2009). We, therefore, tested increased amounts of several media components but were unable to identify any mediumrelated limitation (data not shown). Only a reduction in the kanamycin concentration resulted in an increased growth rate, but most probably reduced plasmid maintenance stability or copy number, as revealed by fluorescence quantification experiments using an mCherry reporter derivative of pMis1_1B ( Figure A1). Mutations in the rep-gene region were already successfully used to increase the copy number of pBBR-based broad host range plasmids and the growth rate of corresponding transformants (Mi et al., 2016;Tao et al., 2005). A more directed screening of rep gene expression level variants might identify a more suitable expression level associated with less plasmid burden.

| Characterization of pMis1_1B as pCM coexpression vector
Despite the lower growth rates of the cotransformants, pMis1_1B is nevertheless a promising candidate for simultaneous gene expression from two plasmids in M. extorquens AM1. To investigate the plasmid stability and general expression levels from pMis1_1B, we used pMis1_P mxaF _mCherry containing the reporter gene under the control of the strong native constitutive promotor P mxaF . Expression of mCherry did not change the growth behavior of the respective strains F I G U R E 3 Investigation of phenotypes for Methylorubrum extorquens AM1 strains carrying pCM160 and pMis1_1B_P mxaF _mCherry. (a) Growth and mCherry fluorescence signal of M. extorquens AM1 containing pCM160, pMis1_1B_P mxaF _mCherry, or both plasmids in combination. Three independent biological replicates were investigated in a microbioreactor system. Colored areas indicate the standard deviation (SD). (b) Ratio of relative PCN of pMis1_1B_P mxaF _mCherry in M. extorquens AM1 + pMis1_1B_P mxaF _mCherry + pCM80 to the same plasmid in M. extorquens AM1 + pMis1_1B_P mxaF _mCherry. The chromosomal housekeeping gene was used as a reference. The mean value of relative pMis1_1B_P mxaF _mCherry PCN of the control strain (= 1) is shown by a dashed line. For each strain, three independent transformants (I-III) were measured.
( Figure A2). We investigated the mCherry expression levels from respective pCM and pMis1_1B constructs in strains with one or two plasmids, respectively. The previously described growth defect of the cotransformants was also visible in this experiment (Figure 3a).
However, there was a clear synergistic effect in terms of expression: The mCherry signal originating from pMis1_1B_P mxaF _mCherry was substantially increased if pCM80 was additionally present. The maximal mCherry fluorescence signal of the cotransformants even exceeded the values from pCM160_mCherry single transformants.
One explanation for this finding could be an increased PCN. We, therefore, determined the relative PCN of pMis1_1B_mCherry for strains containing two plasmids and strains containing only a single plasmid by real-time PCR. Since the efficiency of the used primer pairs varied from 1.73 to 1.96, the E-corrected Pfaffl-method was used (Pfaffl, 2004). Although the values determined for each of the three replicate strains containing two plasmids showed a strong variation, a clear increase in PCN was detectable when both plasmids are present in M. extorquens AM1 (Figure 3b). Thus, the presence of pCM80 led to a strong increase in the pMis1_1B copy number.

| CONCLUSIONS
The pBBR1 derivative pMis1_1B, which we characterized in this study represents a novel plasmid for M. extorquens, which is compatible with the widely

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
The plasmid sequence of pMis1_1B is available in the NCBI GenBank under accession number OP441404: https://www.ncbi.nlm.nih.gov/ nuccore/OP441404. The plasmid itself has been deposited to Addgene. All other data generated or analyzed during this study are included in this published article.

ETHICS STATEMENT
None required.

ORCID
Markus Buchhaupt http://orcid.org/0000-0003-2720-5973 APPENDIX F I G U R E A1 Effect of variation of kanamycin concentration on the growth of Methylorubrum extorquens AM1 pCM80/pMis1_1B_P mxaF _ mCherry cotransformants. The maximal mCherry fluorescence signal is a measure of pMis1_1B_ P mxaF _mCherry abundance, while the maximal slope of the scattered light signal is an indicator of growth rate. Three independent biological replicates were measured; standard deviations are indicated by error bars.
F I G U R E A2 Comparison of scattered light measurements of Methylorubrum extorquens AM1 cotransformants containing pCM80 combined with either pMis1_1B or pMis1_1B_P mxaF _ mCherry. Measurements of biological triplicates are shown.